Polyacrylamide gel electrophoresis (PAGE) is a useful technique for analyzing biological macromolecules such as proteins or nucleic acids. PAGE separates biological molecules based on their electrophoretic mobility, which is a function of the charge, size, and conformation of the molecule. Biological molecules can be analyzed in their native conformation or denatured such that the molecule's mobility through the gel matrix is a function of its length (i.e., size) and its mass-to-charge ratio. A common denaturant for proteins is sodium dodecyl sulfate (SDS), an anionic detergent that linearizes and adds a negative charge to the protein molecule. When SDS is added to the protein sample for gel electrophoresis, the technique is referred to as SDS-PAGE. During PAGE, the sample containing the biological molecule(s) (e.g., analyte) of interest is loaded into a sample well at one end of the gel, and an electric filed is applied across the gel, resulting in the negatively charged analytes migrating from the cathode (negatively charged electrode) towards the anode (positively charged electrode). A dye is typically added to the gel in order to monitor the progress of electrophoresis, and visible or stain free molecular weight markers can be added to one or more sample wells to further monitor the separation of molecules by size. The electric field is removed after the user determines the analyte of interest has migrated through the gel enough to be sufficiently separated from other analytes.
If the molecule of interest is a protein, PAGE separation of the protein analyte can be followed by western analysis. In western analysis, protein analytes are typically transferred to a solid support, such as a filter or membrane (e.g., nitrocellulose or polyvinylidene difluoride (PVDF)). The proteins can be transferred to the membrane by capillary action or electroblotting. In electroblotting, the proteins in the gel are contacted with an electric field that causes the (negatively) charged proteins to migrate out of the gel toward an electrode (anode) and contact the membrane. After the protein of interest is immobilized on the surface of the membrane, the protein is contacted with an antibody (the primary antibody) that specifically binds the protein of interest. The bound antibody is then detected, for example by contacting the primary antibody with a secondary antibody conjugated to a detectable label such as biotin, HRP or AP, or a fluorescent label. The detectable label is then visualized, for example by chemiluminescence. Western analysis provides additional confirmation that a protein or analyte of interest is present in the sample loaded on the gel.